This project focuses on herpesvirus capsid structure and the processes of DNA packaging and capsid completion. Molecular genetics and cryo-electron microscopy (cryoEM) are combined in a tight collaboration to obtain high resolution models that reveal the organization of capsid subunits in situ and particularly the essential minor proteins that interact with the capsid during and following DNA packaging. The locations of most of these essential minor proteins are not known nor are details of their interactions with each other and the capsid. Parallels with dsDNA bacteriophages suggest that the process of translocating the dsDNA chromosome into the herpesvirus capsid is powered by a packaging motor located at the unique portal vertex of the icosahedral capsid and that after the last DNA end has entered the capsid, the portal is closed, and the capsid is stabilized by addition of head completion proteins. Mutant capsids incorporating specifically labeled subunits will be visualized by cryoEM to identify the locations of subunits and to constrain models of subunit fold that may be inferred from density maps. Experiments are divided between two aims. Aim 1 exploits icosahedral symmetry to extend the resolution of cryoEM reconstructions to 5 [unreadable]ngstroms or better, from which elements of subunit folds and interfaces can be determined. Aim 2 abandons icosahedral symmetry to image the unique portal vertex and the DNA packaging proteins that interact with it. Alignment of the portals will involve labeling the constituent UL6 protein, or a bound terminase subunit such as the UL28 protein, to identify the portal's location on each capsid imaged. These portal vertices can then be aligned for calculating reconstructions that dispense with icosahedral symmetry. These Aims both involve significant efforts in optimizing particle preparation and handling along with improving cryoEM imaging and image analysis to collect large datasets of high quality images. Modeling of subunit folds, particularly the essential minor proteins, will rely on direct interpretation of the density maps, fitting of homologous atomic resolution structures from phage capsids, and localization of surface peptides by labeling to constrain and qualify models. The knowledge obtained from these studies enables not only a significantly better understanding of herpesvirus capsid structure, but also provides the means to reveal aspects of how the viral DNA packaging machinery interacts with the capsid during and after DNA packaging. In addition, the essential minor proteins offer novel and highly specific structural targets for the development of antivirals. This proposal will, for example, inform efforts to interfere with assembly, such as by revealing subunit interfaces that may be targeted to inhibit binding. PUBLIC HEALTH RELEVANCE: Herpesviruses directly impact human health, causing chicken pox to cold and genital sores, amongst other diseases. Extending knowledge of the herpesvirus structural proteins and the processes of capsid assembly will significantly aid in the development of highly specific anti-viral drugs to counter herpesvirus infections. This project is highly relevant to NIH's mission by laying the groundwork on which therapeutic remedy may be designed and tested.